The objective of this research plan is a molecular characterization of genetic recombination processes in cultured mammalian cells. For these studies we will use an animal virus, SV40, which most likely depends on host cell enzymes for its recombination. Classical approaches to analysis of recombination with SV40 (and animal viruses in general) are limited in usefulness because of low frequencies of recombination. However, by constructing mixed oligomeric SV40 DNAs in vitro, we apparently have bypassed the rate-limiting step in normal recombination with SV40 as evidenced by a 100 to 500 fold increase in measured recombination frequencies. Because SV40 DNA and these presumptive recombination intermediates are readily accessible to biochemical manipulation in vitro, we can use this sensitive genetic system to investigate molecular details of mitotic recombination in mammalian cells. By a series of two-factor crosses we propose to determine the recombinational properties of monomeric linear DNAs with a variety of end configurations. By enzymatic modification of dimeric linear DNA we propose to determine whether circularization with its attendant possibilities for supercoiling is a prerequisite for recombination. By a series of two-factor crosses using specially constructed partial dimers and three-factor crosses with oligomeric DNA, we propose to measure the average size of heteroduplex segments that link recombined duplexes together. In addition we propose to develop a cell-free recombination system for the longer range goal of defining enzymatic activities and metabolites that are required for these recombination processes. There is at present no other higher eucaryotic system (except of course for other papovaviruses) in which genetic and biochemical approaches can be combined so advantageously to dissect genetic recombination processes in mitotic cells.